Small Molecule Cocktail Can Reactivate the Fragile X Gene in Cells: Progress Report

With a $180,000 grant from FRAXA Research Foundation and The Pierce Family Fragile X Foundation from 2016-2018, Dr. Jeannie Lee and her team at Harvard University and Massachusetts General Hospital have run a series of studies aimed at reactivating the gene, FMR1, which is silenced in Fragile X syndrome. Using state-of-the-art tools, Dr. Lee and colleagues have found a method using combinations of specific drugs which spur the FMR1 gene to produce its normal protein product.

by Jeannie Lee, MD, PhD

Fragile X syndrome (FXS) is caused by expansion of CGG trinucleotide repeats in the FMR1 gene. The Fragile X “full mutation” – a very long expansion resulting in over 200 CGG repeats – causes DNA hyper-methylation and silencing of the FMR1 gene. Defining the mechanism of repeat expansion and gene silencing could pave the way for treatment through FMR1 reactivation.

With funding from FRAXA Research Foundation and The Pierce Family Fragile X Foundation, we have made progress towards this goal. We have discovered a method that will reactivate the FMR1 gene by treating cells with a cocktail of small molecules, under a specific regimen in vitro (patent filing pending). Reactivation levels are significant at both the RNA and protein levels.

In human induced pluripotent stem (iPS) and embryonic stem (ES) cell lines from patients with Fragile X syndrome, the FMR1 gene is usually completely silenced. In our lab, we demonstrated de-repressed FMR1 gene transcription and an increase in the protein FMRP.

FMRP reactivation - Jeannie Lee

Reactivation of FMRP protein from silenced FXS cells, using a cocktail of small molecules.

FMRP levels in the wild type cells and human embryonic stem (ES) cells harboring FXS full mutation grown in regular media (Control) and media with small molecules are measured by western blot. Wildtype samples are loaded with serial dilution (1/3x) for quantitative estimation. GAPDH is an unrelated protein which serves as a loading control.

Next Steps

We are currently following up on this promising observation in several ways:

  1.  We are repeating the experiment using different patient cell lines to rule out clonal and other technical artifacts.
  2.  We are testing whether a similar reactivation can be observed in patient-specific neuronal cells.
  3.  We are investigating the underlying mechanism to better define the reactivation process.

This project shows that we can reactivate a gene in single cells in the lab. Many issues remain to be understood; for example, it is not yet known if this strategy works with all cells or just a small subset of cells (clonal artifact). Much remains to be done. However, this is just the kind of work that must be done to make a definitive cure for Fragile X to be a reality one day. Our ultimate goal is to translate this knowledge and advance this clinical candidate as a FXS treatment mechanism.

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